Expression of ecdysteroid-regulated genes is reduced specifically in the wing discs of the wing-deficient mutant (fl) of Bombyx mori

Abstract

The wing-deficient mutant, flügellos (fl), of the silkworm, lacks four wings in the pupa and the adult. Previous studies have suggested that the fl wing discs lose responsiveness to ecdysteroid during metamorphosis. To test this hypothesis at the molecular level we compared the expression of 12 genes when the wing discs from the wild-type (WT) and fl larvae were cultured in the presence or absence of 20-hydroxyecdysone (20E). Most of the genes tested here, ecdysteroid-inducible (EcR-A, -B1, and E75) and noninducible genes (actin A3, beta-tubulin, apterous (ap), USP, and BHR38) were normally expressed in the fl wing discs. However, the amounts of mRNAs of two ecdysteroid-inducible genes, BHR3 (early-late gene; Bombyx homologue to DHR3 and MHR3) and Urbain (wing-specific late gene), were reduced to about 50% and 20% of WT in the cultured fl wing discs, respectively. We analyzed developmental profiles of these mRNAs during metamorphosis. They also demonstrated decreased BHR3 and Urbain mRNA 2 days after the onset of wandering. This reduction in transcription of BHR3 in the fl mutant was observed only in the wing disc, not in the testis and fatbody. These results imply that the aberrant expression of the fl gene affects the downstream pathway of ecdysteroid signaling specifically in the wing discs and thus leads to a deficiency in wing formation.

CYP306A1, a Cytochrome P450 Enzyme, Is Essential for Ecdysteroid Biosynthesis in the Prothoracic Glands of Bombyx and Drosophila

“…Specific primers for Cyp306a1-Bm (Upper, 5Ј-GACCCAACGGCA-CTGTATATGAGAG-3Ј; Downer, 5Ј-GCCCACTGCAATGGGATCAC-3Ј), sad-Bm (Upper, 5Ј-TAGAGCTCAAAGTGG-3Ј; Downer, 5Ј-TGTCG-TATCTCCAGCGGCG-3Ј), and rpL3 (Upper, 5Ј-CGTCGTCATCGTGG-TAAGGTCAAG-3Ј; Downer, 5Ј-GGTCTCAATGTATCCAACAACACCG-ACAC-3Ј) were used, giving 343-, 412-, and 219-bp fragments, respectively. rpL3 was used as a loading control as described previously (17). 35 cycles (95°C for 10 s, 68°C for 20 s) were carried out for amplification of these genes.…”

Neverland is an evolutionally conserved Rieske-domain protein that is essential for ecdysone synthesis and insect growth

“…Quantitative RT-PCR was performed as described . rpL3 and rp49 were used as loading controls for nvd-Bm and nvd-Dm, respectively (Foley et al, 1993;Matsuoka and Fujiwara, 2000).…”

Positional Cloning of a Bombyx Wingless Locus flügellos (fl) Reveals a Crucial Role for fringe That Is Specific for Wing Morphogenesis

“…Moreover, fl wing discs cultured in medium containing 20-hydroxyecdysone (20E) do not develop, whereas the WT wing discs differentiate normally under the same conditions . During the prepupal stage, the transcription of two ecdysteroid-induced genes, BHR3 (early-late gene; Bombyx homolog of DHR3) and Urbain (wing-specific late gene), is blocked in the fl k wing disc in a tissuespecific manner (Matsuoka and Fujiwara 2000), and the expression of an ecdysteroid-induced 41-kDa protein is also reduced . These observations suggest that fl wing discs cannot respond to ecdysteroid during metamorphosis in the same way as WT wing discs do.…”